MicroRNA: a primer for the cell biologist
1 Nov 2011 by Evoluted New Media
Active in the control of cell functionality at a genetic level, through the regulation of messenger RNA transcription, miRNA is now recognised to be a powerful medium for understanding how cell control pathways function and interact
MicroRNA (miRNA) consists of short ribonucleic acid (RNA) sequences (oligonucleotides), typically only 17-24 nucleotides in length. These sequences were first identified in the early 1990s1 but their role as distinct biological regulators of cell function was not recognised until 2001-2003.
miRNA strands essentially function as post-transcriptional regulators, which bind to matching sequences on target messenger RNA transcripts (mRNAs) resulting in translational suppression and gene silencing (negative regulation). The human genome alone is known to encode for around 1,400 miRNA sequences3 and around 900-1,000 of these have discrete gene regulation functions in around 60% of human genes4.
It has been postulated that miRNA is a major control structure inside cells, acting like a cellular master switch in gene regulation for most processes. This relationship between miRNAs and the regulation of cell function makes them useful as informative behavioural markers. An miRNA profile can provide improved understanding of what is happening within the cell system. Consequently there is great potential in understanding disease processes via miRNA and developing appropriate applications at a cellular level. As a result, three major commercial areas that exploit the properties of miRNA have come to the fore during the period since its discovery and characterisation. These are therapeutic, diagnostic and analytic miRNA tools.
The first area of interest is in the provision of new therapeutic targets for previously untreatable or difficult to treat disease areas; a topic of focus for such companies as Regulus, Miragen, Mirna Therapeutics and RXi Pharmaceuticals. Some miRNAs function as oncogenes and their overexpression contributes to tumourgenesis. Others act as tumour suppressors, which when down-regulated contribute to the development of cancer. The interrelationship between miRNAs is therefore an important factor in understanding, quantifying and treating disease at a cellular level.
Identification of miRNA active in controlling specific cell pathways related to disease processes, could lead to potential new drug therapies if these miRNAs could be blocked. Regulus therapeutics, for example, is an miRNA developer utilising a ‘toolbox’ of oligonucleotides to modulate specific miRNA targets and thus treat the disease areas associated with them.
Another future therapeutic target is diabetes. A pre-clinical study in mice indicated that the antagonism of miR-103 and miR-107 with proprietary chemically modified anti-miRNA oligonucleotides could promote insulin signalling in both liver and adipose tissue5. Overall the study demonstrated that silencing miR-103/107 in animal models of obesity improved glucose homeostasis and insulin sensitivity. The identification of miRNA-103 and miRNA-107 (miR-103/107) in mice as being involved in diabetes has highlighted a potential high-value therapy area5.
Diagnostics is another area where miRNA is quickly becoming useful. For example, specific miRNA tests have now been developed to distinguish between different cell types involved in certain cancers, using miRNA extracted from bodily fluids such as serum, urine or saliva.
miRNA is a major control structure inside cells, acting like a cellular master switch in gene regulation for most processesRosetta Genomics, a molecular diagnostics company, has developed a single miRNA test that can distinguish between different cell types in lung tumours. The test, which uses specific miRNA biomarkers, confers the ability to differentiate between squamous and non-squamous non-small cell lung cancer and also between adenocarcinoma and mesothelioma. In addition and more importantly Rosetta is also developing a miRNA system to identify the origin of tumours in patients presenting with cancer of unknown primary (CUP).
The third area of interest is in cell analytics, and it is here that the full potential of miRNA can be realised.
miRNA profiling can be an enormously powerful technique with wide application. On a basic level, if a cell in an assay is treated with a chemical compound (i.e. a chemical stimulus) there will be a response, and it is the quantification and understanding of that response that allows a researcher to recognise if the chemical applied to the cell is a viable treatment for a disease process; how effective it is; and, if it is active in ways applicable to other diseases.
Conventional cell-based assays are usually developed to be very simple and may only give limited, but still extremely valuable, information based on the response of a single biomarker (for example the presence or concentration of a particular enzyme). miRNA profiling and analysis has the potential to screen a single compound and determine all of its potential effects on a specific cell type. In effect, a single assay can provide a comprehensive ‘dossier’ on a compound’s biological activity.
The ability to extract RNA from cells following chemical exposure and obtain a full miRNA fingerprint using customised miRNA microarray technology is an enormously powerful technique, which has both qualitative and quantitative applications. The combination of miRNA profile data in conjunction with powerful bioinformatic and statistical techniques is set to form the future of this technology and will hopefully build a useful knowledge base on biological processes.
In a novel analytical approach called SistemRNA, Sistemic Ltd has introduced the concept of key microRNA (kmiRs) to the miRNA arena. This has provided the ability to easily understand miRNA data and rapidly integrate it into an existing workflow.
In any cellular process there will be certain miRNAs which are the most relevant and these are identified as the kmiRs. Using this new concept, the study of a cellular process can be simplified to the response of just a few ‘key’ miRNAs instead of having to analyse the whole dataset each time. By applying complex econometric algorithms that can identify emerging patterns (clusters) in miRNA data, the new approach quickly and cost-effectively delivers results in context relating only to kmiRs, making the routine application of miRNA profiling far more realistic for discovery, process and quality control purposes.
An extensive library of compound kmiR profiles for the major therapeutic areas (oncology, inflammation, metabolic disorders, HDACi and lipid metabolism) has been compiled by Sistemic, which can be used to compare against the miRNA profile of unknown compounds to position or reposition biologic compounds in their own or related therapy areas.
A study conducted by Sistemic into a specific cancer cell line provides a good example of how unknown compounds can be rapidly screened for very specific activity using the kmiR concept6.
In this case the cells were treated with four different classes of oncology drug (anti-metabolite, DNA replication inhibitor, epigenetic modifier and statin). The total RNA extract of the cell was initially processed and then subjected to a customised miRNA microarray platform. Using the data from this initial array, a specialised algorithm was used to compare and identify the key miRNAs (kmiRs) clusters against the SistemKB database. The kmiR profile obtained was easily able to identify, define and differentiate between each of the different drugs, as well as provide further data on drug sub-classes. In subsequent studies, data describing only these kmiRs need then be compared in order to establish and identify significant change in the biological system.
The use of miRNA as the basis of commercial analytical technology has now become a reality. Central to cell function at a genetic level, miRNA provides direct access to fundamental cell control mechanisms. Used as an analytical tool miRNA has the power to generate a huge amount of data that can make it difficult to extract salient information. However, a new analytical approach that defines and focuses on what is referred to as key microRNA, or kmiRs, greatly simplifies the application of miRNA studies making them both easier to apply and more practical for routine analyses of cell systems.
miRNA key facts
- miRNAs are extremely discriminating biomarkers that fully describe how cells respond to their environment.
- There are only 1,400 miRNAs in the human genome
- Much of the published data about miRNA relates to identification of the function of each type
- There is still much to learn about:
- The molecular mechanisms by which miRNAs regulate gene expression
- The control relationship between each miRNA i.e., how many mRNAs do they regulate
- How multiple miRNAs interact in the regulation of mRNAs
- Lee, RC; Feinbaum, RL; Ambros, V. The C. Elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell (1993) 75 (5): 843–54.
- Lagos-Quintana M, Rauhut R, Lendeckel W, Tushi T. Identification of novel genes coding for small expressed RNA’s. Science (2001) 294, 853-858.
- Wataru Yoshioka, Wataru Higashiyama and Chiharu Tohyama. Involvement of microRNAs in dioxin-induced liver damage in the mouse, Toxicol. Sci. (2011), doi: 10.1093/toxsci/kfr130. http://toxsci.oxfordjournals.org/content/early/2011/05/20/toxsci.kfr130.abstract
- Robin C. Friedman, Kyle Kai-How Farh, Christopher B. Burge and David P. Bartel. Most mammalian mRNAs are conserved targets of microRNAs, Genome Research (2009), 19 , 92-105. http://genome.cshlp.org/content/19/1/92.full.pdf+html
- Mirko Trajkovski, Jean Hausser and Markus Stoffel et al. MicroRNAs 103 and 107 regulate insulin sensitivity. Nature (2011), 474, 649-653. http://www.nature.com/nature/journal/v474/n7353/full/nature10112.html
- In-house study. microRNA expression utility in Therapeutic Positioning. June 2010, Therapeutic Positioning 10.1; Extending the Range of miRNA Technology. Genetic Engineering and Biotechnology News (GEN), November 15, 2010, Vol. 30, No. 20, http://www.genengnews.com/gen-articles/extending-the-range-of-mirna-technology/3476/ accessed 14th July 2011
Contact t: +44 (0) 141 330 1683 f: + 44 (0) 141 330 2062 e: verna.mcerlane@sistemic.co.uk w: www.sistemic.co.uk
